Acrosome reaction of fowl sperm: evidence for shedding of the acrosomal cap in intact form to release acrosomal enzyme.

The aim of this study was to determine the site of enzyme release from the acrosome and the fate of the acrosomal cap during the process of acrosome reaction (AR) in fowl sperm. Gelatin substrate coverslips with halos were subjected to scanning electron microscopy to determine the site from which acrosomal proteolytic enzyme was released to form a halo around the acrosome of individual sperm. Aliquots of sperm treated with solubilized inner perivitelline layer (IPL) containing 5 mmol CaCl(2) were simultaneously subjected to fluorescent staining with fluorescein isothiocyanate-labeled peanut agglutinin and scanning electron microscopy to evaluate AR of sperm and to examine the status of the acrosomal region, respectively. Inside the halos, a gelatin-free (proteolyzed gelatin) layer was found extending some distance around the acrosome of sperm. All of the sperm showing the formation of halos on gelatin had a single circular opening around their subacrosomal rod at the base of the acrosomal cap. Interaction of sperm with solubilized IPL in the presence of 5 mmol CaCl(2) resulted in 41.4 ± 1.8% of the sperm to undergo AR, as evaluated by fluorescein isothiocyanate-labeled peanut agglutinin. Similarly, as observed using scanning electron microscopy, 38.2 ± 2.3% of the sperm treated with solubilized IPL plus 5 mmol CaCl(2) had exposed subacrosomal rod. In all sperm examined, no sign of disruption of the acrosomal membrane was found in the apical region of the acrosome. Rather, the acrosomal caps were found intact detached from the acrosomal region of the sperm, indicating that AR of fowl sperm resulted in the intact removal of the acrosomal cap. Based on these experimental observations, we suggest that the process of AR in fowl sperm is unique; the release of the acrosomal proteolytic enzyme may occur through a single circular opening formed at the base of the acrosomal cap and the acrosomal cap is detached in intact form from the posterior acrosomal region of the sperm.

Plumping fluid added to storage medium increases twofold the functional life span of fowl spermatozoa in vitro at 4°C.

1. The objective of this study was to examine whether addition of plumping fluid (PF) to Lake's solution (LS) for storage of fowl spermatozoa in vitro at 4°C can prolong survival and improve the quality of spermatozoa. 2. In experiment 1, aliquots of spermatozoa were stored in vitro in LS alone and LS containing 10%, 25%, 50% and 75% (v:v) PF for 0.5, 24, 48, 72, 96 and 120 h at 4°C. After the end of each storage period, spermatozoa were evaluated for their viability, mobility and penetrability. Viability was determined using SYBR-14 and propidium iodide (PI) staining. Mobility was assessed using an Accudenz assay. Penetrability was assessed using spermatozoa-inner perivitelline layer (IPL) interaction assay. 3. In experiment 2, aliquots of spermatozoa were stored in vitro in LS alone and LS containing 25% and 50% (v:v) PF for 0.5, 24, 48 and 72 h at 4°C, and then fertility of the spermatozoa was evaluated using intravaginal artificial insemination (AI) in hens. 4. Storage of spermatozoa in LS alone resulted in loss of viability, mobility, penetrability and fertility within 48 h. In contrast, no loss of viability and penetrability was observed for the spermatozoa stored for 48, 96, 72 and 48 h in LS containing 10%, 25%, 50% and 75% (v:v) PF, respectively. In particular, fertilising capacity was not lost for the spermatozoa stored in the presence of 25% or 50% PF in LS for 48 and 24 h, respectively. 5. In conclusion, these findings demonstrated that in vitro exposure of fowl spermatozoa to PF during hypothermic storage in LS prolonged spermatozoa survival. A 25% (v:v) level of inclusion of PF in LS may be effective for the improvement of viability, penetrability and fertilising ability of the stored spermatozoa.

Sesquiterpene progenitor, germacrene A: an alarm pheromone in aphids.

Germacrene A, the elusive biogenetic "parent" of many sesquiterpenes, has been isolated from the spotted alfalfa aphid and identified as a new intrageneric aphid alarm pheromone.

Action of the novel antioxidants 4GBE43 and 2BBE43 against lipid peroxidation.

The action and the effect of the newly synthesized compounds 4GBE43 [N-(1,2-diethyltetrahydro-1H-pyrazol-4-yl)-4-[(2E)-3,7-diethyl-2,6-octadienyl] oxybenzamide] and 2BBE43 [2-(benzyloxy)-N-(1,2-diethyltetrahydro-1H-pyrazol-4-yl)benzamide] against lipid peroxidation were studied. 4GBE43 and 2BBE43 quenched the ESR signal of diphenylpicrylhydrazyl (DPPH), suggesting that 4GBE43 and 2BBE43 act as scavengers of free radicals and that each compound quenched 6 free radical molecules. These compounds suppressed the oxidation of methyl linoleate emulsions and soybean phosphatidylcholine liposomes by a free radical initiator, suggesting that these compounds quench the lipid peroxyl radical. 4GBE43 and 2BBE43 also suppressed the spontaneous oxidation of rat brain homogenates. The inhibitory effect of 2BBE43 was of the same order of magnitude (IC50) as that of probucol. The IC50 of 4GBE43 was on the same order of magnitude as that of alpha-tocopherol. However, 4GBE43 at 10(-4)-10(-5) M completely inhibited peroxidation, showing it to be more effective than alpha-tocopherol. These results suggest that 4GBE43 and 2BBE43 act as antioxidants by quenching the lipid peroxyl radical.

Actinoplanones A and B, new cytotoxic polycyclic xanthones from Actinoplanes sp.

Two new cytotoxic polycyclic xanthones, actinoplanones A (1) and B (2) were isolated from the culture broth of Actinoplanes sp. R-304 by monitoring their bioactivity against HeLa cells. Compound 1 was extremely cytotoxic (IC50 0.00004 micrograms/ml) against HeLa cells. The structures of 1 and 2 were established mainly by analyses of 2D heteronuclear correlation NMR experiments. The absolute configurations of the asymmetric carbons of the compounds have been assigned to be 9R, 24S, 25R and 27S by circular dichroism spectra and NMR analysis using chiral derivatives (esters of alpha-methoxy-alpha-(trifluoromethyl)acetic acid).

Actinoplanones C, D, E, F and G, new cytotoxic polycyclic xanthones from Actinoplanes sp.

Our previous finding of strong cytotoxic polycyclic xanthones, actinoplanones A (1) and B (2), in the culture broth of Actinoplanes sp. R-304 stimulated us to isolate further five new cytotoxic polycyclic xanthones which were named actinoplanones C (3), D (4), E (5), F (6) and G (7) from the broth. Actinoplanones C (3) and G (7) showed very strong cytotoxicity against HeLa cells at less than 0.00004 microgram/ml dosage (IC50). The structures of 3-7 were varieties of 1 for the N-2 and C-4 substituents. All or several actinoplanones showed strong antimicrobial activities against bacteria and the rice blast fungus. Actinoplanone A (1) was tested for cytotoxicity against various tumor cells and for inhibitory effect on HeLa cell macromolecular synthesis, and 1 exhibited strong cytotoxicity against the cells and inhibitory action on DNA synthesis.

Maturational changes in motility, acrosomal proteolytic activity, and penetrability of the inner perivitelline layer of fowl sperm, during their passage through the male genital tract.

The objective was to examine, in vitro, the motility, acrosomal proteolytic activity (APA), and penetrating ability of fowl sperm recovered from the testis and epididymis, as well as the proximal, middle, and distal vas deferens, to assess the potential fertilizing ability of sperm as a function of maturation. A motile sperm separation technique was used to estimate sperm motility with Accudenz, a gelatin slide technique was used to measure the diameter of the halo around the acrosome of individual sperm as an indication of APA, and a sperm-inner perivitelline layer (IPL) interaction assay was done to estimate the number of hole formations as an indication of sperm penetration into the IPL. Sperm in the testis exhibited the least motility, produced the smallest halos, and created the least number of holes per 0.25 mm(2). Motility, diameter of the halo, and number of holes increased gradually (P < 0.05) from the epididymis to the distal vas deferens and were markedly different (P < 0.05) between testicular and deferent duct sperm. Based on these in vitro experimental findings, we inferred that fowl sperm undergo a gradual process of maturational changes in motility, APA, and penetrability as a means of acquiring potential fertility during their passage throughout the male genital tract.

Maturational changes in the survivability and fertility of fowl sperm during their passage through the male reproductive tract.

The objective of this study was to examine whether domestic fowl (Gallus domesticus) sperm undergo maturation in their capacity for survival and fertilization in the male reproductive tract. Sperm collected from the testis, epididymis and the proximal, middle and distal vas deferens were simultaneously stored in vitro in minimum essential medium (MEM) at 39°C for 0, 3 and 6h, and at 4°C for 24 and 48h. Sperm membrane integrity was measured using the dual fluorescent stain SYBR-14/propidium iodide (PI). Aliquots of sperm from the various sites were subjected to artificial insemination (AI) into the uteri of hens to assess the duration of sperm survival in the oviduct and to determine the fertility status of the sperm. Testicular sperm exhibited a very low capacity to survive under in vitro liquid storage conditions, irrespective of the storage temperature used, and in the oviduct, and they had a low ability to fertilize the ovum. On the contrary, sperm from the distal vas deferens had a higher survival rate during in vitro storage periods, a longer life span in the oviduct, and high fertility. Survival and fertilizing capacity of the sperm recovered from the testes increased gradually (P<0.05) from the testes to the distal vas deferens. In conclusion, we suggest that fowl sperm may undergo functional maturation through a process of gradual changes in their survival and fertilization capacities during their passage through the successive parts of the male reproductive tract.

Olfactory receptor responses of the nymphal American cockroach to sex pheromones and their mimics.

1. EAG responses to highly purified sex pheromones (periplanone-A and -B), sex pheromone mimics [germacrene-D, (+)-verbanyl acetate and (+)-trans-verbenyl acetate] and general odor (camphor) were recorded from both sexes of adult and three nymphal stages (7, 10 and 11th instars) of the American cockroach. 2. The M/F ratios were evaluated for each stage by stimulation with the above chemicals. 3. The ratio values indicated undeveloped sex pheromone receptor on the antennae of 7th male instar and females of all the stages. On the other hand, precursory development of the receptor was expected on the antennae of males of the old-aged nymphal instars and full development on the adult male antennae.

Sex pheromonal activity of (+)-bornyl acetate and related compounds to the American cockroach.

Many compounds related to (+)-bornyl acetate (a sex phero-mone mimic of the American cockroach) were synthesized and tested for sex pheromonal activity. All compounds except for esters of (+)- and (-)-borneol were inactive, whereas (+)-bornyl acetate (Ib) and propionate (Ic) showed the activity at 0.05 mg. Although (-)-bornyl propionate (IVc) is the enantiomer of Ic, it exhibited weak activity at 0.5 mg dose. On the basis of the behavioral assay results, important chemical factors in Ib for pheromonal activity were elucidated and are discussed in connection with another mimic, (+)-trans-verbenyl acetate. The M/ F ratio index in EAG was evaluated for both active and inactive compounds. The index demonstrated a good correlation with the behavioral activity.

Sex pheromonal activity of (+)-trans-verbenyl acetate and related compounds to the american cockroach,Periplaneta americana L.

The sex pheromone mimic of the American cockroach, (+)-trans-verbenyl acetate, and 67 synthesized analogs were tested for sex pheromonal activity using males of the cockroach. Only three esters in addition to the original (+)-trans-verbenyl acetate exhibited activity. (+)-trans-Verbenyl propionate and (+)-verbanyl acetate in particular showed stronger activity than the original mimic. None of the other analogs caused a response at the 1-mg level. The lower threshold levels at which even the most active analogs showed activity (0.02 mg) were many orders of magnitude higher than that of the natural pheromone, periplanone-B (10(-8) mg). From the structure-activity relationships, important structural factors for sex pheromonal activity in the original mimic were discussed.

Structural significance of the alkyl substituent at the C-4 of (+)-trans-verbenyl acetate in sex pheromonal activity of the American cockroach.

The significance of the alkyl group at the C-4 of (+)-trans-verbenyl acetate, which is the sex pheromone mimic of the American cockroach, was investigated. Seven alcohols possessing an ethyl, propyl, or dimethyl group at this position of the 6,6-dimethylbicyclo[3.1.1]heptane skeleton were synthesized and evaluated by behavioral assay. All of the alcohols were inactive, while three of four acetates of the 2α-alcohols induced sexual behavior in male cockroaches at the 0.02 or 0.5 mg dosage level, either of which is many orders of magnitude higher than the threshold level of the natural sex pheromones (10(-8) mg). Among the acetates, the compounds with a methyl group or an α-oriented ethyl group at C-4 showed the highest activity. The results are discussed in terms of spatial requirements of the molecules for interactions with the receptor.

Studies on relationship between activity and electron density on carbonyl oxygen in sex pheromone mimics of the American cockroach, part XI.

From the relationship between structures of the 2α-substituents of verbanyl analogs and their sex pheromone activities to the American cockroach, electron density of the carbonyl oxygen atom in the substituent, as estimated by the [(17)O]NMR chemical shift, was estimated to be an important factor which influenced the activity, in addition to length of the substituent and the position of the carbonyl group. (+)-Verbanyl methylcarbonate (XX), possessing the highest electron density on the carbonyl oxygen atom, showed the strongest activity among the analogs.

Structural significance of the geminal-dimethyl group of (+)-trans-verbenyl acetate, sex pheromone mimic of the American cockroach.

The significance of the geminal dimethyl group at C-6 of (+)-trans-veibenyl acetate (Ib), a sex pheromone mimic of the American cockroach, was investigated. Our interest was first directed to eliminating either methyl group of the geminal dimethyl group. Consequently, alcohols (IIIa and IVa) possessing a monomethyl group at C-6 were successfully obtained. Acetate (IVb) and propionate (IVc) of alcohol (IVa) possessing only a C-9 methyl group induced sex pheromonal activity at the 0.5 (IVb) and 0.02 mg (IVc) dose levels, respectively, while acetate (IIIb) of a alcohol (IIIa) with only C-8 methyl group was inactive. Alcohol (Va), which has an ethyl group at the C-6 instead of C-9 methyl, was also synthesized and converted into its acetate (Vb), which showed no activity. From these results, the significance of the dimethyl group is discussed relative to spatial requirements of the molecule for the receptor space. The M/F ratio in EAG was also evaluated for the synthesized esters. The M/F ratio index represented well the potency of pheromonal activity.

Studies on the etiologic mechanism of anisakiasis. --1. Immunological reactions of digestive tract induced by Anisakis larva.

In an attempt to determine the etiological mechanism of anisakiasis, immunological studies were performed using guinea pigs and rabbits. In order to carry out these examinations, we used a specific antigen, hemoglobin of Anisakis larva. We demonstrate that the etiological mechanism of anisakiasis involves anaphylactic reaction as well as Arthus reaction in the digestive tract. Cell-mediated immune reaction also have to consider the relation of establishment of allergic condition in experimental anisakiasis. These results suggest that the three types allergic reaction may play the main role in clinical symptomes of human anisakiasis.

Studies on the etiologic mechanism of anisakiasis. --2. Epidemiologic study of inhabitants and questionaire survey in Japan.

A nationwide investigation on the incidence of anisakiasis over a period of 5 years from 1973 through to 1977 was conducted by questionaire sent to 851 main medical centers throughout Japan. The number of cases for all Japanese for a period of 5 years were 334 cases of gastric anisakiasis and 45 intestinal anisakiasis. We investigated both a random group of 3,108 inhabitants of Hokkaido and 42 anisakiasis patients using skin test, indirect hemagglutination test and measurement of serum IgE. We have demonstrated that the etiologic mechanism of acute anisakiasis involved anaphylactic reaction as well as Arthus reaction in the digestive tract.

Inhibitory effect of flavonoids on DNA-dependent DNA and RNA polymerases.

Flavonoids, (-)-epigallocatechin (1), myricetin (2) and quercetin (3), were investigated for inhibitory effects on E. coli DNA polymerase I and T7 bacteriophage RNA polymerase. In both DNA and RNA synthesis, 1 and 3 inhibited enzyme reactions by non-competitive and mixed type inhibition respectively, with regard to template DNAs. Myricetin (2) inhibited DNA and RNA polymerase reactions by mixed type and competitive type inhibition, respectively, with template DNAs. It was suggested that 2 interacts with covalently closed circular DNA.

Effect of pisiferic acid and its derivatives on cytotoxicity macromolecular synthesis and DNA polymerase alpha of HeLa cells.

1. Seventy-seven derivatives of pisiferic acid (2), an antimicrobial diterpenoid, were tested for cytotoxicity against HeLa cells and 9 derivatives were found to show cytotoxicity at less than 2 micrograms/ml dose (IC50). 2. Hydrophobicity was revealed to be an important factor for cytotoxicity of the derivatives. 3. Compound 2 inhibited predominantly DNA synthesis in HeLa cells as compared with RNA and protein synthesis. 4. No direct interaction between 2 and nucleic acid bases was indicated by a u.v. spectral method. 5. Several of the pisiferic acid species showed inhibitory action on HeLa DNA polymerase alpha, and the inhibitory activity was about 1/20 of aphidicolin.